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Nutritional quality of protein in the leaves of eleven Asphodeline species (Liliaceae) from Turkey
Food Chemistry 135 (2012) 1360–1364
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Nutritional quality of protein in the leaves of eleven Asphodeline species (Liliaceae) from Turkey Gokhan Zengin a, Abdurrahman Aktumsek a, Gokalp-Ozmen Guler b, Yavuz-Selim Cakmak a, Julio Girón-Calle c, Manuel Alaiz c, Javier Vioque c,⇑ a b c
Selcuk University, Science Faculty, Department of Biology, 42075 Konya, Turkey Selcuk University, Ahmet Kelesoglu Education Faculty, Department of Biological Education, 42090 Konya, Turkey Instituto de la Grasa, C.S.I.C. Avda Padre García tejero 4, 41012 Sevilla, Spain
a r t i c l e
i n f o
Article history: Received 25 November 2011 Received in revised form 7 May 2012 Accepted 22 May 2012 Available online 31 May 2012 Keywords: Asphodeline Leaf protein Amino acids Nutritional quality
a b s t r a c t The nutritional quality of the protein in the leaves of 11 Asphodeline (Liliaceae) species was investigated by the determination of the amino acid composition and calculation of several nutritional parameters. The average protein content was 4.7% and ranged from 2.5% in Asphodeline damascena ssp. rugosa to 8.2% in A. turcica. The most abundant essential amino acids were Thr (5.7%), Val (6.0%), Ile (4.7%), and Trp (2.1%). The amino acid composition of Asphodeline peshmeniana was well equilibrated according to Food and Agriculture Organisation standards, but Lys and sulphur amino acids were at limiting concentrations in all the other taxa. Determination of the protein efficiency ratio and biological value revealed that the protein in the leaves of Asphodeline species is of high nutritional quality. Hence, the Asphodeline leaves that are typically used in Turkey for the preparation of salads, represent a good source of protein with high levels of several essential amino acids and a good nutritional value. Analysis of the similarity based on the amino acid composition indicated the existence of different clusters that are consistent with the taxonomical classification, area of distribution, and morphological similarities of the Asphodeline species. Ó 2012 Elsevier Ltd. All rights reserved.
1. Introduction Many vegetables are used for the preparation of salads, including the green leaves of lettuce, spinach, and Brassica spp. The leaves of other lesser known vegetables, for instance Eruca, Nasturtium, and Diplotaxis spp., are consumed in more limited geographical areas. From a nutritional point of view, the consumption of leafy vegetables is recommended as a source of functional components such as polyphenols, glucosinolates (in Brassica spp.), and dietary fibre. In addition, green leaves are between 5% and 10% protein, and may represent a significant cholesterol-free source of protein in the diet. The extractability, solubility, and nutritional value of leaf proteins have been studied (Fiorentini, & Galoppini, 1981), and the use of leaf protein concentrates as food complements in order to provide additional protein in the diet has been suggested (Dale, Allen, Laser, & Lynd, 2009). The genus Asphodeline includes 14 species and belongs to the Liliaceae family together with other genera of great economical and nutritional importance such as Allium. It is present in southwest of Asia, mostly in Middle-Eastern countries, and in the Mediterranean region. This taxon is represented in Turkey by 20 ⇑ Corresponding author. Tel.: +34 954611550; fax: +34 9546167990. E-mail address: [email protected] (J. Vioque). 0308-8146/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.foodchem.2012.05.084
species, 11 of which are endemic to this country (Mathews & Tuzlaci, 1984; Tuzlaci, 1987). Asphodeline species are abundant in Turkey especially in the mountains and steppes of inner Anatolia. Because of the high number of endemisms, this country is considered to be the centre of origin of the genus (Tuzlaci, 1987). Several of the turkish Asphodeline species, including Asphodeline cilicica, A. damascena, Asphodeline globifera, Asphodeline lutea, and Asphodeline taurica, are consumed in salads. The aims of this work were to evaluate the nutritional quality of the protein in the leaves of 11 Asphodeline species distributed throughout Turkey through the determination of the amino acid composition, and several nutritional parameters. The final aim was to determine which of these species could be of interest from a nutritional point of view. In addition, data on the amino acid composition have been used for cluster analysis of the Asphodeline taxa included in this work. 2. Material and methods 2.1. Materials Diethyl ethoxymethylenemanolate was purchased from Fluka. All other chemicals were of analytical grade. Samples of Asphodeline leaves were taken from wild populations in Turkey (Table 1). Healthy green leaves were taken from the basal leaves rosette of
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G. Zengin et al. / Food Chemistry 135 (2012) 1360–1364 Table 1 Collection sites of Asphodeline taxa. Species
Location
Altitude (m)
A. anatolica Tuzlaci (En)
Isparta: between Sarkikaraagac and Yenisarbademli, 38° 030 0700 N, 31° 170 5100 E Adana: between Catalan and Aladag, 37° 270 3700 N, 35° 200 1200 E Kayseri: Yaylali, Zinc mine road, 38° 000 3300 N, 35° 230 5500 E Konya: between Konya and Afyon, 38° 020 51.600 N, 32° 260 4000 E Kayseri: between Yahyali and Sazak, 38° 050 1800 N, 35° 210 3800 E Konya: between Cevizli and Beysehir, 37° 100 27.300 N, 31 ° 480 0800 E Kahramanmaras: Göksun, between Ericek and Karadut, 38° 050 0500 N, 36° 530 4900 E Nigde: Camardi, Mazmili Mount, 37° 390 5500 N, 35° 040 4500 E Konya: between Konya and Beysehir, beside Altinapa Dam Lake, 37° 530 5000 N, 32° 180 2800 E Antalya: Gazipasa, Ciglik village, Asarbasi, 36° 200 2100 N, 32° 310 4700 E Sivas: between Sivas and Bingol village, 39° 430 3800 N, 37° 060 0800 E Antalya: Gebiz, Sanlibel, 37° 200 0100 N, 31° 010 4500 E
1140
A. cilicica Tuzlaci (En) A. globifera J. Gay ex Baker A. damascena (Boiss.) Baker subsp. damascena (Boiss.) Baker A. damascena (Boiss.) Baker subsp. rugosa Tuzlaci (En) A. lutea (L) Reichb. A. peshmeniana Tuzlaci (En) A. prismatocarpa J. Gay ex Baker (En) A. rigidifolia (Boiss.) Baker (En)
A. sertachae Tuzlaci (En) A. taurica (Pallas) Kunth A. turcica Tuzlaci (En)
1080 1170 1170 1210 1200 1420 1970 1270
1610 1380 1300
En: endemic to Turkey.
several plants in a population. Voucher specimens of these populations are deposited at KNYA Herbarium at the Department of Biology, Selcuk University, Konya, Turkey. 2.2. Amino acid analysis Dry leaves were blended with a domestic blender. The resulting flour was used for the analysis of leaf proteins amino acids composition. Samples were hydrolyzed by incubation in 6 N HCl at 110 °C for 24 h. The amino acids were determined after derivatization with diethyl ethoxymethylenemanolate by high-performance liquid chromatography (HPLC), according to the method described by Alaiz, Navarro, Girón, and Vioque (1992), using D, La-aminobutyric acid as internal standard. The HPLC system (Beckman-Coulter) consisted of a 126 solvent module, 166 detector and IBM personal computer. Data acquisition and processing were effected with 32 Karat 7.0 version software (Beckman-Coulter). Samples (20 lL) were injected in a reversed-phase column (Novapack C18, 300 3.9 mm i.d., 4 lm; Waters). Tryptophan was determined by HPLC-RP chromatography after basic hydrolysis according to Yust et al., 2004. Protein content was based on amino acid analysis (Hidalgo, Alaiz, & Zamora, 2001). 2.3. Determination of nutritional parameters The amino acid composition of Asphodeline samples was used for determination of several nutritional parameters as follows: – Amino acid score (chemical score): % essential amino acids in sample/% essential amino acid recommended by FAO (FAO/WHO/UNU, 1985). – Protein efficiency ratio (PER) was calculated according to the following three equations (Alsmeyer, Cunningham, & Happich, 1974): PER1 = 0.684 + 0.456 Leu 0.047 Pro.
PER2 = 0.468 + 0.454 Leu 0.105 Tyr. PER3 = 1.816 + 0.435 Met + 0.78 Leu + 0.211 Hys 0.944 Tyr. – Predicted biological value (BV) was calculated according to Morup, and Olesen (1976) using the following equation: BV = 102.15 Lys0.41 (Phe + Tyr)0.60 (Met + Cys)0.77 Thr0.24 Trp0.21. where each amino acid symbol represents: % amino acid/% amino acid FAO pattern (1985), if % amino acid 6 % amino acid FAO pattern, or: % amino acid FAO pattern (1985)/% amino acid, if % amino acid P % amino acid FAO pattern. 2.4. Cluster analysis Cluster analysis of Asphodeline species was performed using the furthest neighbour method based on an euclidean distance matrix in the Stagraphics 5.1 software. 3. Results and discussion The protein content of the Asphodeline leaves ranged from 2.5% in A. damascena ssp. rugosa to 8.2% in A. turcica (Table 2). The average protein content was 4.7%, which is consistent with previous reports on protein content in the leaves of higher plants (Rao & Polacchi, 1972). The nutritional quality of food proteins depends mainly on their amino acid composition. As shown in Table 2, the amino acid composition of the leaves in all the Asphodeline taxa that were included in this study is very similar. It has been suggested that the amino acid composition of the leaves of most terrestrial plants is very similar due to the very low variability in the amino acid composition of ribulose 1,5-biphospahte carboxylase/oxygenase (rubisco), which accounts for a very large portion of the total leaf protein (Byers, 1983). The composition in essential amino acids of all the Asphodeline species were above the FAO recommendations (FAO/WHO/UNU,
5.8
2.8 1.1 6.6
6.3b 3.5 2.5c
1.4
10.6 ± 0.1 24.0 ± 0.2 5.1 ± 0.0 2.7 ± 0.1 5.2 ± 0.1 5.1 ± 0.1 4.7 ± 0.1 6.9 ± 0.0 1.1 ± 0.0 2.3 ± 0.0 5.8 ± 0.0 0.7 ± 0.1 0.8 ± 0.0 4.2 ± 0.0 2.5 ± 0.2 8.0 ± 0.0 5.2 ± 0.0 5.0 ± 0.0 8.2 ± 0.1 10.1 ± 0.1 19.4 ± 0.1 6.0 ± 0.1 2.1 ± 0.2 5.6 ± 0.1 5.7 ± 0.2 3.9 ± 0.0 7.6 ± 0.0 1.8 ± 0.1 2.2 ± 0.0 6.4 ± 0.0 0.8 ± 0.1 0.9 ± 0.0 5.1 ± 0.0 2.2 ± 0.1 9.4 ± 0.1 6.1 ± 0.0 4.7 ± 0.0 5.2 ± 0.0 12.3 ± 1.1 22.0 ± 0.6 5.8 ± 0.0 2.8 ± 0.3 5.7 ± 0.2 5.9 ± 0.2 4.1 ± 0.1 7.1 ± 0.0 0.9 ± 0.2 2.4 ± 0.2 5.6 ± 0.1 0.5 ± 0.2 1.0 ± 0.2 4.1 ± 0.1 2.3 ± 0.1 8.0 ± 0.2 4.6 ± 0.0 4.9 ± 0.2 6.0 ± 0.2 11.4 ± 0.6 16.1 ± 0.4 6.3 ± 0.1 2.1 ± 0.3 6.3 ± 0.2 6.3 ± 0.1 4.9 ± 0.1 8.8 ± 0.1 1.4 ± 0.1 2.5 ± 0.0 5.7 ± 0.0 0.7 ± 0.1 0.7 ± 0.0 4.8 ± 0.1 1.3 ± 0.2 9.4 ± 0.1 5.9 ± 0.1 5.6 ± 01 3.5 ± 0.1 10.2 ± 0.3 17.0 ± 0.7 6.0 ± 0.1 2.3 ± 0.0 6.6 ± 0.0 5.1 ± 0.2 4.7 ± 0.0 8.1 ± 0.4 1.4 ± 0.1 2.4 ± 0.1 5.2 ± 0.0 1.0 ± 0.3 1.0 ± 0.0 5.4 ± 0.1 1.4 ± 0.1 10.0 ± 0.1 6.7 ± 0.0 5.5 ± 0.2 4.3 ± 0.1 10.5 ± 1.2 15.4 ± 0.2 6.5 ± 0.4 2.5 ± 0.0 6.8 ± 0.0 5.8 ± 0.1 6.7 ± 0.5 7.1 ± 0.4 2.1 ± 0.1 2.9 ± 0.1 6.0 ± 0.5 1.5 ± 0.1 1.2 ± 0.1 4.4 ± 0.1 1.0 ± 0.2 8.3 ± 0.1 5.3 ± 0.0 6.1 ± 0.3 2.9 ± 0.3
c
a
b
Suggested pattern of amino acid meeting FAO requirements (FAO/WHO/UNU, 1985). Tyr + Phe. Met + Cys.
9.3 ± 0.2 19.4 ± 0.4 6.2 ± 0.0 2.1 ± 0.2 5.5 ± 0.1 6.4 ± 0.2 3.7 ± 0.0 8.2 ± 0.1 1.7 ± 0.1 2.6 ± 0.1 6.4 ± 0.3 0.8 ± 0.0 1.0 ± 0.0 5.1 ± 0.1 2.5 ± 0.1 8.8 ± 0.1 5.6 ± 0.1 4.8 ± 0.1 4.2 ± 0.1 10.5 ± 0.1 16.9 ± 0.3 6.3 ± 0.0 2.1 ± 0.1 7.2 ± 0.1 6.0 ± 0.3 4.2 ± 0.1 7.4 ± 0.0 1.7 ± 0.2 2.3 ± 0.3 5.6 ± 0.1 0.9 ± 0.2 1.0 ± 0.0 4.7 ± 0.0 3.2 ± 0.1 9.3 ± 0.1 5.2 ± 0.0 5.6 ± 0.0 2.5 ± 0.0 9.7 ± 0.3 20.0 ± 0.1 6.0 ± 0.0 2.5 ± 0.1 6.1 ± 0.2 6.0 ± 0.2 4.0 ± 0.0 7.4 ± 0.1 2.3 ± 0.1 2.0 ± 0.1 5.9 ± 0.0 1.1 ± 0.1 1.1 ± 0.0 4.7 ± 0.0 2.3 ± 0.1 8.8 ± 0.1 5.0 ± 0.0 5.1 ± 0.0 4.3 ± 0.2 10.0 ± 0.6 19.6 ± 0.1 5.5 ± 0.4 2.5 ± 0.0 6.5 ± 0.0 5.5 ± 0.1 4.4 ± 0.1 8.3 ± 0.0 0.7 ± 0.1 2.3 ± 0.0 5.0 ± 0.0 1.0 ± 0.0 0.9 ± 0.0 5.1 ± 0.0 2.4 ± 0.0 9.2 ± 0.1 5.6 ± 0.0 5.5 ± 0.1 6.5 ± 0.1 11.7 ± 0.0 23.7 ± 0.1 5.6 ± 0.1 2.7 ± 0.1 6.3 ± 0.6 4.8 ± 0.1 3.8 ± 0.0 7.4 ± 0.2 0.9 ± 0.1 1.7 ± 0.2 6.8 ± 0.2 1.1 ± 0.1 0.9 ± 0.0 4.3 ± 0.0 2.0 ± 0.1 7.3 ± 0.0 4.3 ± 0.1 4.7 ± 0.0 3.5 ± 0.2 10.6 ± 0.3 18.3 ± 0.2 5.8 ± 0.1 2.5 ± 0.0 6.5 ± 0.0 5.7 ± 0.2 4.6 ± 0.1 7.8 ± 0.1 1.0 ± 0.1 2.1 ± 0.0 7.0 ± 0.5 1.0 ± 0.4 1.0 ± 0.0 4.8 ± 0.0 2.0 ± 0.2 8.6 ± 0.0 5.4 ± 0.0 5.4 ± 0.0 5.1 ± 0.1 Asp + Asn Glu + Gln Ser His Gly Thr Arg Ala Pro Tyr Val Met Cys Ile Trp Leu Phe Lys Protein
A. turcica A. taurica A. sertachae A. rigidifolia A. prismatocarpa A. peshmeniana A. lutea A. damascena ssp. rugosa A. damascena ssp damascena A. globifera A. cilicica A. anatolica
Table 2 Amino acid composition and protein content in the leaves of Asphodeline species. Data, g /100 g protein, are the average ± standard error of two determinations.
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G. Zengin et al. / Food Chemistry 135 (2012) 1360–1364
FAOa
1362
1985) except for Lys and sulphur amino acids. The most abundant essential amino acids as compared to FAO recommendations are Thr, with an average value of 5.7% and a FAO recommendation of 1.4%, followed by Val (6.0%, FAO recommendation 3.5%), Leu (4.7%, FAO recommendation 2.8%), and Trp (2.1%, FAO recommendation 1.1%). A. peshmeniana was the only species that met all FAO recommendations concerning essential amino acids. This is in contrast with previous reports describing that the content of Lys in leaves exceeds the FAO recommendations (Byers, 1971). The amino acid score, indicating the percentage of essential amino acids as compared to the FAO recommendations, are shown in Table 3. This score represents a simplified model for predicting the dietary protein quality, although it does not take into account the amino acid digestibility and availability (Moughan, 2005; Sarwar et al., 1984). The highest amino acid scores were found in A. damascena ssp. rugosa and A. lutea. The latter species are very common components of salads in Turkey. The amino acid scores and the essential to total amino acid ratios, which are also shown in Table 3, are high in Asphodeline due to the elevated content in Thr, Val, Ile, Trp, and Leu. These amino acid scores and essential to total amino acid ratios were higher than those observed by our previous studies in pulses, such as Vicia faba (Vioque, Alaiz, & Girón-Calle, 2012), Lupinus spp. (Pastor-Cavada, Juan, Pastor, Alaiz, & Vioque, 2009), Lathyrus spp. (Pastor-Cavada, Juan, Pastor, Alaiz, & Vioque, 2011a) and Vicia spp. (Pastor-Cavada, Juan, Pastor, Alaiz, & Vioque, 2011b). A number of parameters that are based on the amino acid composition have been developed in order to estimate the overall nutritional quality of proteins, which depends on several factors in addition to the composition in essential amino acids. These include theoretical protein efficiency ratios and theoretical biological values. The protein efficiency ratio (PER) is calculated by the determination of the weight gain divided by the protein intake, and is a basic parameter of protein nutritional quality. Nevertheless, theoretical PER values, calculated as described in materials and methods bear a good relationship with experimental PER values, and are also a very good indicator of protein quality (Alsmeyer et al., 1974). PER values lower than 1.5 and higher than 2 indicate low and high quality proteins, respectively (Friedman, 1996). The theoretical PER values of Asphodeline leaf proteins were higher than three in most taxa, including species traditionally eaten in salads, such as A. damascena and A. taurica. A. peshmeniana has the highest PER value (Table 3). Asphodeline PER values were even higher than those observed in protein crops widely consumed, such as soybean (2.57) (Wolzak, Elias, & Bressani, 1981), chickpea (2.8) (Newman, Roth, Newman, & Lockerman, 1987), rice (1.98) and wheat (1.59). (Wolzak et al., 1981). While high PER1, PER2 and PER3 values were due to high Leu content, high PER2 and PER3 were due to low Tyr content. Although Asphodeline is low in the aromatic amino acid Tyr, the Tyr plus Phe value is above the FAO recommendations for aromatic amino acids. The biological value is an experimental determination of the proportion of ingested proteins that are incorporated into the organism of test animals. The predicted biological values for the Asphodeline species, which were determined as described in materials and methods using the amino acid composition data, ranged between 49.8 in A. turcica and 77.0 in A. peshmeniana (Table 3). These values were high mainly because of the high contents of Thr. Asphodeline biological values, which were higher than those reported for pulses such as V. faba (40.2) (Vioque et al., 2012) and Lathyrus sp. (24.5–55.4) (Pastor-Cavada et al., 2011a), and similar to those observed in cereals, such as wheat (61.6) and triticale (65.3) (Friedman,1996). Because of a favourable amino acid composition as shown by the theoretical PER values and the predicted biological values, the nutritional quality of the protein in Asphodeline leaves is higher
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G. Zengin et al. / Food Chemistry 135 (2012) 1360–1364 Table 3 Nutritional characteristics of Asphodeline leaf protein.
%EAA/TAAa AASb BVc PER1d PER2 PER3 a b c d
A. anatolica
A. cilicica
A. globifera
A. damascena ssp. damascena
A. damascena ssp. rugosa
A. lutea
A. peshmeniana
A. prismatocarpa
A. rigidifolia
A. sertachae
A. taurica
A. turcica
45.5 142.6 68.4 3.2 3.2 3.9
40.6 127.3 72.9 2.6 2.7 3.3
45.0 141.1 59.7 3.5 3.5 4.2
44.5 139.5 70.1 3.2 3.3 4.2
45.9 143.9 57.9 3.5 3.5 4.1
45.9 143.9 50.5 3.3 3.3 3.4
45.0 141.1 77.0 3.0 3.0 3.1
46.0 144.2 64.5 3.8 3.8 4.6
45.0 141.1 51.1 3.5 3.5 3.9
42.1 132.0 51.9 2.9 2.9 3.0
45.6 143.0 50.5 3.5 3.5 4.2
42.3 132.6 49.8 2.9 2.9 3.1
Essential amino acids/total amino acids. Amino acid score. Biological value. Protein efficiency ratio.
than the quality of protein in the seeds of other wild Mediterranean plants such as Lathyrus (Pastor-Cavada et al., 2011a) and Vicia (Pastor-Cavada et al., 2011b), which have also been proposed as alternative sources of food protein. This amino acid composition is very similar to the composition of Rubisco in Thr, Val, Ile, Trp, and Leu, reflecting the fact that Rubisco is the mayor protein component in leaves. Data on amino acid composition has also been used to carry out a cluster analysis of the Asphodeline species. This is not the first time that the leaf protein amino acid composition has been used in taxonomy (Yeoh, & Watson, 1982). The dendrogram that was generated by cluster analysis (Fig. 1) shows a certain correlation with the taxonomic relationships within species belonging to the Asphodeline genus that were previously established considering
100
D 80
60
C
40
A
B
the morphological characters (Tuzlaci, 1987). Thus, cluster A includes the two subspecies of A. damascena that have been included in the present study. Cluster B includes A. lutea and A. taurica, two perennial species that usually grow in the same habitat and share similar morphological characteristics, i.e. leafy stems and simple inflorescences. Cluster C includes two taxonomically related species, A. globifera and A. rigidifolia. Cluster D includes species that grow in the southern region of Turkey, two of which were recently described, Asphodeline sertachae and A. turcica (Tuzlaci, 1998). Finally, A. peshmeniana which could not be ascribed to any of the clusters, is characterised by being the taxa that is able to grow in a greater variety of habitats. In conclusion, the protein in the leaves of the Asphodeline species included in this investigation is characterised in general by a good nutritional quality as determined by comparison of amino acid composition with FAO recommendations, although it is for most taxa deficient in sulphur amino acids and Lys. This is a limitation that can be overcome by complementation with other protein sources in the diet, as occurs for instance with protein from seed legumes, which is characteristically low in sulphur amino acids, and benefits from being consumed together with cereals that are rich in these amino acids. Nevertheless, one of the taxa, A. peshmeniana, met all the FAO recommendations concerning the amino acid composition, including the content in sulphur amino acids and Lys. Nutritional parameters were also high and above those reported for the seed proteins of other Mediterranean edible plants. Thus, the leaves of Asphodeline as a main ingredient of salads, which is a typical use of these plants in different regions of Turkey, represent a good source of proteins of good nutritional value in addition to functional compounds, such as polyphenols and dietary fibre.
20 Acknowledgements
A. turcica
A. sertachae
A. cilicica
A. peshmeniana
A. rigidifolia
A. prismatocarpa
A. globifera
A. taurica
A. lutea
A. damascena ssp. rugosa
A. damascena ssp. damascena
A. anatolica
0
Fig. 1. Dendogram produced by cluster analysis of the amino acid composition of the protein in Asphodeline leaves (Table 2).
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Food Chemistry journal homepage: www.elsevier.com/locate/foodchem
Short communication
Nutritional quality of protein in the leaves of eleven Asphodeline species (Liliaceae) from Turkey Gokhan Zengin a, Abdurrahman Aktumsek a, Gokalp-Ozmen Guler b, Yavuz-Selim Cakmak a, Julio Girón-Calle c, Manuel Alaiz c, Javier Vioque c,⇑ a b c
Selcuk University, Science Faculty, Department of Biology, 42075 Konya, Turkey Selcuk University, Ahmet Kelesoglu Education Faculty, Department of Biological Education, 42090 Konya, Turkey Instituto de la Grasa, C.S.I.C. Avda Padre García tejero 4, 41012 Sevilla, Spain
a r t i c l e
i n f o
Article history: Received 25 November 2011 Received in revised form 7 May 2012 Accepted 22 May 2012 Available online 31 May 2012 Keywords: Asphodeline Leaf protein Amino acids Nutritional quality
a b s t r a c t The nutritional quality of the protein in the leaves of 11 Asphodeline (Liliaceae) species was investigated by the determination of the amino acid composition and calculation of several nutritional parameters. The average protein content was 4.7% and ranged from 2.5% in Asphodeline damascena ssp. rugosa to 8.2% in A. turcica. The most abundant essential amino acids were Thr (5.7%), Val (6.0%), Ile (4.7%), and Trp (2.1%). The amino acid composition of Asphodeline peshmeniana was well equilibrated according to Food and Agriculture Organisation standards, but Lys and sulphur amino acids were at limiting concentrations in all the other taxa. Determination of the protein efficiency ratio and biological value revealed that the protein in the leaves of Asphodeline species is of high nutritional quality. Hence, the Asphodeline leaves that are typically used in Turkey for the preparation of salads, represent a good source of protein with high levels of several essential amino acids and a good nutritional value. Analysis of the similarity based on the amino acid composition indicated the existence of different clusters that are consistent with the taxonomical classification, area of distribution, and morphological similarities of the Asphodeline species. Ó 2012 Elsevier Ltd. All rights reserved.
1. Introduction Many vegetables are used for the preparation of salads, including the green leaves of lettuce, spinach, and Brassica spp. The leaves of other lesser known vegetables, for instance Eruca, Nasturtium, and Diplotaxis spp., are consumed in more limited geographical areas. From a nutritional point of view, the consumption of leafy vegetables is recommended as a source of functional components such as polyphenols, glucosinolates (in Brassica spp.), and dietary fibre. In addition, green leaves are between 5% and 10% protein, and may represent a significant cholesterol-free source of protein in the diet. The extractability, solubility, and nutritional value of leaf proteins have been studied (Fiorentini, & Galoppini, 1981), and the use of leaf protein concentrates as food complements in order to provide additional protein in the diet has been suggested (Dale, Allen, Laser, & Lynd, 2009). The genus Asphodeline includes 14 species and belongs to the Liliaceae family together with other genera of great economical and nutritional importance such as Allium. It is present in southwest of Asia, mostly in Middle-Eastern countries, and in the Mediterranean region. This taxon is represented in Turkey by 20 ⇑ Corresponding author. Tel.: +34 954611550; fax: +34 9546167990. E-mail address: [email protected] (J. Vioque). 0308-8146/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.foodchem.2012.05.084
species, 11 of which are endemic to this country (Mathews & Tuzlaci, 1984; Tuzlaci, 1987). Asphodeline species are abundant in Turkey especially in the mountains and steppes of inner Anatolia. Because of the high number of endemisms, this country is considered to be the centre of origin of the genus (Tuzlaci, 1987). Several of the turkish Asphodeline species, including Asphodeline cilicica, A. damascena, Asphodeline globifera, Asphodeline lutea, and Asphodeline taurica, are consumed in salads. The aims of this work were to evaluate the nutritional quality of the protein in the leaves of 11 Asphodeline species distributed throughout Turkey through the determination of the amino acid composition, and several nutritional parameters. The final aim was to determine which of these species could be of interest from a nutritional point of view. In addition, data on the amino acid composition have been used for cluster analysis of the Asphodeline taxa included in this work. 2. Material and methods 2.1. Materials Diethyl ethoxymethylenemanolate was purchased from Fluka. All other chemicals were of analytical grade. Samples of Asphodeline leaves were taken from wild populations in Turkey (Table 1). Healthy green leaves were taken from the basal leaves rosette of
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G. Zengin et al. / Food Chemistry 135 (2012) 1360–1364 Table 1 Collection sites of Asphodeline taxa. Species
Location
Altitude (m)
A. anatolica Tuzlaci (En)
Isparta: between Sarkikaraagac and Yenisarbademli, 38° 030 0700 N, 31° 170 5100 E Adana: between Catalan and Aladag, 37° 270 3700 N, 35° 200 1200 E Kayseri: Yaylali, Zinc mine road, 38° 000 3300 N, 35° 230 5500 E Konya: between Konya and Afyon, 38° 020 51.600 N, 32° 260 4000 E Kayseri: between Yahyali and Sazak, 38° 050 1800 N, 35° 210 3800 E Konya: between Cevizli and Beysehir, 37° 100 27.300 N, 31 ° 480 0800 E Kahramanmaras: Göksun, between Ericek and Karadut, 38° 050 0500 N, 36° 530 4900 E Nigde: Camardi, Mazmili Mount, 37° 390 5500 N, 35° 040 4500 E Konya: between Konya and Beysehir, beside Altinapa Dam Lake, 37° 530 5000 N, 32° 180 2800 E Antalya: Gazipasa, Ciglik village, Asarbasi, 36° 200 2100 N, 32° 310 4700 E Sivas: between Sivas and Bingol village, 39° 430 3800 N, 37° 060 0800 E Antalya: Gebiz, Sanlibel, 37° 200 0100 N, 31° 010 4500 E
1140
A. cilicica Tuzlaci (En) A. globifera J. Gay ex Baker A. damascena (Boiss.) Baker subsp. damascena (Boiss.) Baker A. damascena (Boiss.) Baker subsp. rugosa Tuzlaci (En) A. lutea (L) Reichb. A. peshmeniana Tuzlaci (En) A. prismatocarpa J. Gay ex Baker (En) A. rigidifolia (Boiss.) Baker (En)
A. sertachae Tuzlaci (En) A. taurica (Pallas) Kunth A. turcica Tuzlaci (En)
1080 1170 1170 1210 1200 1420 1970 1270
1610 1380 1300
En: endemic to Turkey.
several plants in a population. Voucher specimens of these populations are deposited at KNYA Herbarium at the Department of Biology, Selcuk University, Konya, Turkey. 2.2. Amino acid analysis Dry leaves were blended with a domestic blender. The resulting flour was used for the analysis of leaf proteins amino acids composition. Samples were hydrolyzed by incubation in 6 N HCl at 110 °C for 24 h. The amino acids were determined after derivatization with diethyl ethoxymethylenemanolate by high-performance liquid chromatography (HPLC), according to the method described by Alaiz, Navarro, Girón, and Vioque (1992), using D, La-aminobutyric acid as internal standard. The HPLC system (Beckman-Coulter) consisted of a 126 solvent module, 166 detector and IBM personal computer. Data acquisition and processing were effected with 32 Karat 7.0 version software (Beckman-Coulter). Samples (20 lL) were injected in a reversed-phase column (Novapack C18, 300 3.9 mm i.d., 4 lm; Waters). Tryptophan was determined by HPLC-RP chromatography after basic hydrolysis according to Yust et al., 2004. Protein content was based on amino acid analysis (Hidalgo, Alaiz, & Zamora, 2001). 2.3. Determination of nutritional parameters The amino acid composition of Asphodeline samples was used for determination of several nutritional parameters as follows: – Amino acid score (chemical score): % essential amino acids in sample/% essential amino acid recommended by FAO (FAO/WHO/UNU, 1985). – Protein efficiency ratio (PER) was calculated according to the following three equations (Alsmeyer, Cunningham, & Happich, 1974): PER1 = 0.684 + 0.456 Leu 0.047 Pro.
PER2 = 0.468 + 0.454 Leu 0.105 Tyr. PER3 = 1.816 + 0.435 Met + 0.78 Leu + 0.211 Hys 0.944 Tyr. – Predicted biological value (BV) was calculated according to Morup, and Olesen (1976) using the following equation: BV = 102.15 Lys0.41 (Phe + Tyr)0.60 (Met + Cys)0.77 Thr0.24 Trp0.21. where each amino acid symbol represents: % amino acid/% amino acid FAO pattern (1985), if % amino acid 6 % amino acid FAO pattern, or: % amino acid FAO pattern (1985)/% amino acid, if % amino acid P % amino acid FAO pattern. 2.4. Cluster analysis Cluster analysis of Asphodeline species was performed using the furthest neighbour method based on an euclidean distance matrix in the Stagraphics 5.1 software. 3. Results and discussion The protein content of the Asphodeline leaves ranged from 2.5% in A. damascena ssp. rugosa to 8.2% in A. turcica (Table 2). The average protein content was 4.7%, which is consistent with previous reports on protein content in the leaves of higher plants (Rao & Polacchi, 1972). The nutritional quality of food proteins depends mainly on their amino acid composition. As shown in Table 2, the amino acid composition of the leaves in all the Asphodeline taxa that were included in this study is very similar. It has been suggested that the amino acid composition of the leaves of most terrestrial plants is very similar due to the very low variability in the amino acid composition of ribulose 1,5-biphospahte carboxylase/oxygenase (rubisco), which accounts for a very large portion of the total leaf protein (Byers, 1983). The composition in essential amino acids of all the Asphodeline species were above the FAO recommendations (FAO/WHO/UNU,
5.8
2.8 1.1 6.6
6.3b 3.5 2.5c
1.4
10.6 ± 0.1 24.0 ± 0.2 5.1 ± 0.0 2.7 ± 0.1 5.2 ± 0.1 5.1 ± 0.1 4.7 ± 0.1 6.9 ± 0.0 1.1 ± 0.0 2.3 ± 0.0 5.8 ± 0.0 0.7 ± 0.1 0.8 ± 0.0 4.2 ± 0.0 2.5 ± 0.2 8.0 ± 0.0 5.2 ± 0.0 5.0 ± 0.0 8.2 ± 0.1 10.1 ± 0.1 19.4 ± 0.1 6.0 ± 0.1 2.1 ± 0.2 5.6 ± 0.1 5.7 ± 0.2 3.9 ± 0.0 7.6 ± 0.0 1.8 ± 0.1 2.2 ± 0.0 6.4 ± 0.0 0.8 ± 0.1 0.9 ± 0.0 5.1 ± 0.0 2.2 ± 0.1 9.4 ± 0.1 6.1 ± 0.0 4.7 ± 0.0 5.2 ± 0.0 12.3 ± 1.1 22.0 ± 0.6 5.8 ± 0.0 2.8 ± 0.3 5.7 ± 0.2 5.9 ± 0.2 4.1 ± 0.1 7.1 ± 0.0 0.9 ± 0.2 2.4 ± 0.2 5.6 ± 0.1 0.5 ± 0.2 1.0 ± 0.2 4.1 ± 0.1 2.3 ± 0.1 8.0 ± 0.2 4.6 ± 0.0 4.9 ± 0.2 6.0 ± 0.2 11.4 ± 0.6 16.1 ± 0.4 6.3 ± 0.1 2.1 ± 0.3 6.3 ± 0.2 6.3 ± 0.1 4.9 ± 0.1 8.8 ± 0.1 1.4 ± 0.1 2.5 ± 0.0 5.7 ± 0.0 0.7 ± 0.1 0.7 ± 0.0 4.8 ± 0.1 1.3 ± 0.2 9.4 ± 0.1 5.9 ± 0.1 5.6 ± 01 3.5 ± 0.1 10.2 ± 0.3 17.0 ± 0.7 6.0 ± 0.1 2.3 ± 0.0 6.6 ± 0.0 5.1 ± 0.2 4.7 ± 0.0 8.1 ± 0.4 1.4 ± 0.1 2.4 ± 0.1 5.2 ± 0.0 1.0 ± 0.3 1.0 ± 0.0 5.4 ± 0.1 1.4 ± 0.1 10.0 ± 0.1 6.7 ± 0.0 5.5 ± 0.2 4.3 ± 0.1 10.5 ± 1.2 15.4 ± 0.2 6.5 ± 0.4 2.5 ± 0.0 6.8 ± 0.0 5.8 ± 0.1 6.7 ± 0.5 7.1 ± 0.4 2.1 ± 0.1 2.9 ± 0.1 6.0 ± 0.5 1.5 ± 0.1 1.2 ± 0.1 4.4 ± 0.1 1.0 ± 0.2 8.3 ± 0.1 5.3 ± 0.0 6.1 ± 0.3 2.9 ± 0.3
c
a
b
Suggested pattern of amino acid meeting FAO requirements (FAO/WHO/UNU, 1985). Tyr + Phe. Met + Cys.
9.3 ± 0.2 19.4 ± 0.4 6.2 ± 0.0 2.1 ± 0.2 5.5 ± 0.1 6.4 ± 0.2 3.7 ± 0.0 8.2 ± 0.1 1.7 ± 0.1 2.6 ± 0.1 6.4 ± 0.3 0.8 ± 0.0 1.0 ± 0.0 5.1 ± 0.1 2.5 ± 0.1 8.8 ± 0.1 5.6 ± 0.1 4.8 ± 0.1 4.2 ± 0.1 10.5 ± 0.1 16.9 ± 0.3 6.3 ± 0.0 2.1 ± 0.1 7.2 ± 0.1 6.0 ± 0.3 4.2 ± 0.1 7.4 ± 0.0 1.7 ± 0.2 2.3 ± 0.3 5.6 ± 0.1 0.9 ± 0.2 1.0 ± 0.0 4.7 ± 0.0 3.2 ± 0.1 9.3 ± 0.1 5.2 ± 0.0 5.6 ± 0.0 2.5 ± 0.0 9.7 ± 0.3 20.0 ± 0.1 6.0 ± 0.0 2.5 ± 0.1 6.1 ± 0.2 6.0 ± 0.2 4.0 ± 0.0 7.4 ± 0.1 2.3 ± 0.1 2.0 ± 0.1 5.9 ± 0.0 1.1 ± 0.1 1.1 ± 0.0 4.7 ± 0.0 2.3 ± 0.1 8.8 ± 0.1 5.0 ± 0.0 5.1 ± 0.0 4.3 ± 0.2 10.0 ± 0.6 19.6 ± 0.1 5.5 ± 0.4 2.5 ± 0.0 6.5 ± 0.0 5.5 ± 0.1 4.4 ± 0.1 8.3 ± 0.0 0.7 ± 0.1 2.3 ± 0.0 5.0 ± 0.0 1.0 ± 0.0 0.9 ± 0.0 5.1 ± 0.0 2.4 ± 0.0 9.2 ± 0.1 5.6 ± 0.0 5.5 ± 0.1 6.5 ± 0.1 11.7 ± 0.0 23.7 ± 0.1 5.6 ± 0.1 2.7 ± 0.1 6.3 ± 0.6 4.8 ± 0.1 3.8 ± 0.0 7.4 ± 0.2 0.9 ± 0.1 1.7 ± 0.2 6.8 ± 0.2 1.1 ± 0.1 0.9 ± 0.0 4.3 ± 0.0 2.0 ± 0.1 7.3 ± 0.0 4.3 ± 0.1 4.7 ± 0.0 3.5 ± 0.2 10.6 ± 0.3 18.3 ± 0.2 5.8 ± 0.1 2.5 ± 0.0 6.5 ± 0.0 5.7 ± 0.2 4.6 ± 0.1 7.8 ± 0.1 1.0 ± 0.1 2.1 ± 0.0 7.0 ± 0.5 1.0 ± 0.4 1.0 ± 0.0 4.8 ± 0.0 2.0 ± 0.2 8.6 ± 0.0 5.4 ± 0.0 5.4 ± 0.0 5.1 ± 0.1 Asp + Asn Glu + Gln Ser His Gly Thr Arg Ala Pro Tyr Val Met Cys Ile Trp Leu Phe Lys Protein
A. turcica A. taurica A. sertachae A. rigidifolia A. prismatocarpa A. peshmeniana A. lutea A. damascena ssp. rugosa A. damascena ssp damascena A. globifera A. cilicica A. anatolica
Table 2 Amino acid composition and protein content in the leaves of Asphodeline species. Data, g /100 g protein, are the average ± standard error of two determinations.
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G. Zengin et al. / Food Chemistry 135 (2012) 1360–1364
FAOa
1362
1985) except for Lys and sulphur amino acids. The most abundant essential amino acids as compared to FAO recommendations are Thr, with an average value of 5.7% and a FAO recommendation of 1.4%, followed by Val (6.0%, FAO recommendation 3.5%), Leu (4.7%, FAO recommendation 2.8%), and Trp (2.1%, FAO recommendation 1.1%). A. peshmeniana was the only species that met all FAO recommendations concerning essential amino acids. This is in contrast with previous reports describing that the content of Lys in leaves exceeds the FAO recommendations (Byers, 1971). The amino acid score, indicating the percentage of essential amino acids as compared to the FAO recommendations, are shown in Table 3. This score represents a simplified model for predicting the dietary protein quality, although it does not take into account the amino acid digestibility and availability (Moughan, 2005; Sarwar et al., 1984). The highest amino acid scores were found in A. damascena ssp. rugosa and A. lutea. The latter species are very common components of salads in Turkey. The amino acid scores and the essential to total amino acid ratios, which are also shown in Table 3, are high in Asphodeline due to the elevated content in Thr, Val, Ile, Trp, and Leu. These amino acid scores and essential to total amino acid ratios were higher than those observed by our previous studies in pulses, such as Vicia faba (Vioque, Alaiz, & Girón-Calle, 2012), Lupinus spp. (Pastor-Cavada, Juan, Pastor, Alaiz, & Vioque, 2009), Lathyrus spp. (Pastor-Cavada, Juan, Pastor, Alaiz, & Vioque, 2011a) and Vicia spp. (Pastor-Cavada, Juan, Pastor, Alaiz, & Vioque, 2011b). A number of parameters that are based on the amino acid composition have been developed in order to estimate the overall nutritional quality of proteins, which depends on several factors in addition to the composition in essential amino acids. These include theoretical protein efficiency ratios and theoretical biological values. The protein efficiency ratio (PER) is calculated by the determination of the weight gain divided by the protein intake, and is a basic parameter of protein nutritional quality. Nevertheless, theoretical PER values, calculated as described in materials and methods bear a good relationship with experimental PER values, and are also a very good indicator of protein quality (Alsmeyer et al., 1974). PER values lower than 1.5 and higher than 2 indicate low and high quality proteins, respectively (Friedman, 1996). The theoretical PER values of Asphodeline leaf proteins were higher than three in most taxa, including species traditionally eaten in salads, such as A. damascena and A. taurica. A. peshmeniana has the highest PER value (Table 3). Asphodeline PER values were even higher than those observed in protein crops widely consumed, such as soybean (2.57) (Wolzak, Elias, & Bressani, 1981), chickpea (2.8) (Newman, Roth, Newman, & Lockerman, 1987), rice (1.98) and wheat (1.59). (Wolzak et al., 1981). While high PER1, PER2 and PER3 values were due to high Leu content, high PER2 and PER3 were due to low Tyr content. Although Asphodeline is low in the aromatic amino acid Tyr, the Tyr plus Phe value is above the FAO recommendations for aromatic amino acids. The biological value is an experimental determination of the proportion of ingested proteins that are incorporated into the organism of test animals. The predicted biological values for the Asphodeline species, which were determined as described in materials and methods using the amino acid composition data, ranged between 49.8 in A. turcica and 77.0 in A. peshmeniana (Table 3). These values were high mainly because of the high contents of Thr. Asphodeline biological values, which were higher than those reported for pulses such as V. faba (40.2) (Vioque et al., 2012) and Lathyrus sp. (24.5–55.4) (Pastor-Cavada et al., 2011a), and similar to those observed in cereals, such as wheat (61.6) and triticale (65.3) (Friedman,1996). Because of a favourable amino acid composition as shown by the theoretical PER values and the predicted biological values, the nutritional quality of the protein in Asphodeline leaves is higher
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G. Zengin et al. / Food Chemistry 135 (2012) 1360–1364 Table 3 Nutritional characteristics of Asphodeline leaf protein.
%EAA/TAAa AASb BVc PER1d PER2 PER3 a b c d
A. anatolica
A. cilicica
A. globifera
A. damascena ssp. damascena
A. damascena ssp. rugosa
A. lutea
A. peshmeniana
A. prismatocarpa
A. rigidifolia
A. sertachae
A. taurica
A. turcica
45.5 142.6 68.4 3.2 3.2 3.9
40.6 127.3 72.9 2.6 2.7 3.3
45.0 141.1 59.7 3.5 3.5 4.2
44.5 139.5 70.1 3.2 3.3 4.2
45.9 143.9 57.9 3.5 3.5 4.1
45.9 143.9 50.5 3.3 3.3 3.4
45.0 141.1 77.0 3.0 3.0 3.1
46.0 144.2 64.5 3.8 3.8 4.6
45.0 141.1 51.1 3.5 3.5 3.9
42.1 132.0 51.9 2.9 2.9 3.0
45.6 143.0 50.5 3.5 3.5 4.2
42.3 132.6 49.8 2.9 2.9 3.1
Essential amino acids/total amino acids. Amino acid score. Biological value. Protein efficiency ratio.
than the quality of protein in the seeds of other wild Mediterranean plants such as Lathyrus (Pastor-Cavada et al., 2011a) and Vicia (Pastor-Cavada et al., 2011b), which have also been proposed as alternative sources of food protein. This amino acid composition is very similar to the composition of Rubisco in Thr, Val, Ile, Trp, and Leu, reflecting the fact that Rubisco is the mayor protein component in leaves. Data on amino acid composition has also been used to carry out a cluster analysis of the Asphodeline species. This is not the first time that the leaf protein amino acid composition has been used in taxonomy (Yeoh, & Watson, 1982). The dendrogram that was generated by cluster analysis (Fig. 1) shows a certain correlation with the taxonomic relationships within species belonging to the Asphodeline genus that were previously established considering
100
D 80
60
C
40
A
B
the morphological characters (Tuzlaci, 1987). Thus, cluster A includes the two subspecies of A. damascena that have been included in the present study. Cluster B includes A. lutea and A. taurica, two perennial species that usually grow in the same habitat and share similar morphological characteristics, i.e. leafy stems and simple inflorescences. Cluster C includes two taxonomically related species, A. globifera and A. rigidifolia. Cluster D includes species that grow in the southern region of Turkey, two of which were recently described, Asphodeline sertachae and A. turcica (Tuzlaci, 1998). Finally, A. peshmeniana which could not be ascribed to any of the clusters, is characterised by being the taxa that is able to grow in a greater variety of habitats. In conclusion, the protein in the leaves of the Asphodeline species included in this investigation is characterised in general by a good nutritional quality as determined by comparison of amino acid composition with FAO recommendations, although it is for most taxa deficient in sulphur amino acids and Lys. This is a limitation that can be overcome by complementation with other protein sources in the diet, as occurs for instance with protein from seed legumes, which is characteristically low in sulphur amino acids, and benefits from being consumed together with cereals that are rich in these amino acids. Nevertheless, one of the taxa, A. peshmeniana, met all the FAO recommendations concerning the amino acid composition, including the content in sulphur amino acids and Lys. Nutritional parameters were also high and above those reported for the seed proteins of other Mediterranean edible plants. Thus, the leaves of Asphodeline as a main ingredient of salads, which is a typical use of these plants in different regions of Turkey, represent a good source of proteins of good nutritional value in addition to functional compounds, such as polyphenols and dietary fibre.
20 Acknowledgements
A. turcica
A. sertachae
A. cilicica
A. peshmeniana
A. rigidifolia
A. prismatocarpa
A. globifera
A. taurica
A. lutea
A. damascena ssp. rugosa
A. damascena ssp. damascena
A. anatolica
0
Fig. 1. Dendogram produced by cluster analysis of the amino acid composition of the protein in Asphodeline leaves (Table 2).
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